title: Discovery and confirmation of O-GlcNAcylated proteins in rat liver mitochondria by combination of mass spectrometry and immunological methods.
abstract: O-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-glycosidic linkage to serine and threonine residues. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid. In this study, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver. First, highly purified mitochondrial proteins were obviously shown to be O-GlcNAcylated by immunoblot profiling. Then, β-elimination followed by Michael Addition with Dithiothreitol (BEMAD) treatment and LC-MS/MS were performed to enrich and identify O-GlcNAcylated mitochondrial proteins, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly, all of these O-GlcNAcylated mitochondrial proteins are enzymes, the majority of which are involved in a wide variety of biological processes, such as urea cycle, tricarboxylic acid cycle and lipid metabolism, indicating a role for protein O-GlcNAcylation in mitochondrial function.
authors.affiliation: Institutes of Biomedical Sciences and Department of Chemistry, Fudan University, Shanghai, China.
authors.forename: Weiqian
authors.initials: W
authors.lastname: Cao
authors.forename: Jing
authors.initials: J
authors.lastname: Cao
authors.forename: Jiangming
authors.initials: J
authors.lastname: Huang
authors.forename: Jun
authors.initials: J
authors.lastname: Yao
authors.forename: Guoquan
authors.initials: G
authors.lastname: Yan
authors.forename: Haoqi
authors.initials: H
authors.lastname: Xu
authors.forename: Pengyuan
authors.initials: P
authors.lastname: Yang
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D006031:Glycosylation; D013058:Mass Spectrometry; D008930:Mitochondria, Liver / Q000378:metabolism*; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000378:metabolism*; D025941:Protein Interaction Mapping; D060066:Protein Interaction Maps; D011499:Protein Processing, Post-Translational; D051381:Rats; D015203:Reproducibility of Results; D015398:Signal Transduction
chemical_list: D024101:Mitochondrial Proteins; D000117:Acetylglucosamine